Presence Of ANA Research Articles

SAT0001 IFN signature is associated with autoantibody profiles in patients with myositis

BackgroundThe IFN signature has been described to be present in many autoimmune diseases, including myositis. The extent of this signature appears to be related to disease activity, however, the underlying...

S(+)MDMA and 5-HT Evoke Dissimilar Ionic Currents in Human Serotonin Transporter-Expressing Cells

Although monoamine transporters translocate substrates (neurotransmitters) across the plasma membrane, a second less studied function is their ion channel-like activity which is responsible for the passive permeation of ions through the membrane. These currents are inward at resting membrane potential and would result in cell depolarization, which may enhance presynaptic excitability, and which would help explain the elevated release of neurotransmitter associated by psychostimulants. Here we compare the ionic currents evoked by serotonin (5-HT) and S(+)-3,4-methylenedioxy-N-methylamphetamine (S(+)MDMA, ecstasy) in mammalian cells expressing the human serotonin transporter (hSERT). 5-HT as well as S(+)MDMA induce inward currents in hSERT-expressing cells. Although the conductances are similar, the S(+)MDMA induced-current reversal potential is strongly shifted towards the Na+ equilibrium potential, indicating a bigger contribution of Na+ to the overall current compared to 5-HT current. To measure changes in Na+ permeability induced by these compounds, the cells were pre-loaded with the Na+ sensor Asante NaTRIUM GREEN 2 and changes in intracellular Na+ concentration ([Na+]i) were monitored fluorometrically. Dose-response experiments show that 5-HT and S(+)MDMA have similar potency (EC50 ∼200 nM) in elevating the [Na+]i; however, S(+)MDMA increases [Na+]i at least 3 times with respect to 5-HT. In addition, the reconstitution of hSERT on a lipid bilayer in the presence of a Na+ gradient showed channel-like activity after the application of 5-HT or S(+)MDMA in the trans side. Taken together these results strongly suggest that S(+)MDMA-induced current is different in composition than the 5-HT current, and moreover it must be strongly depolarizing due to its higher Na+ content.Supported by RC1DA028112 and R01DA033930.

IFN signature is associated with autoantibody profiles in patients with myositis

Backgroundand objectivesThe interferon (IFN) signature has been described to be present in many autoimmune diseases, including myositis. The extent of this signature appears to be related to disease activity, however,...

Cevimeline-Induced Monophasic Salivation from the Mouse Submandibular Gland: Decreased Na+Content in Saliva Results from Specific and Early Activation of Na+/H+Exchange

Cevimeline and pilocarpine are muscarinic agonists used clinically to treat dry mouth. In this study, we explored fluid secretion from mouse submandibular glands to determine the mechanism of cevimeline, pilocarpine, and an experimentally used agent carbachol. Cevimeline evoked almost the same amount of secretion at concentrations from 30 μM to 1 mM. Pilocarpine also induced secretion at a concentration as low as 1 μM and was the most powerful secretagogue at 10 μM. Secretion was induced by carbachol at 0.1 μM, with maximum secretion at 1.0 μM. Cevimeline induced monophasic secretion at all concentrations tested, whereas higher concentrations of pilocarpine and carbachol induced secretion with variable kinetics, i.e., an initial transient high flow rate, followed by decreased secretion after 2 to 3 min. In the presence of an epithelial Na(+) channel blocker, amiloride, neither carbachol nor pilocarpine affected the Na(+) level of secreted saliva; however, it significantly increased the Na(+) content of cevimeline-induced saliva. The intracellular Ca(2+) response of acinar cells was almost identical among all three agents, although recovery after drug removal was slower for cevimeline and pilocarpine. A profound decrease in intracellular pH was observed during pilocarpine and carbachol treatment, whereas intracellular acidification induced by cevimeline was only seen in the presence of a Na(+)/H(+) exchange inhibitor. When external HCO(3)(-) was removed, cevimeline-induced saliva significantly decreased. These findings suggest that cevimeline specifically activates Na(+)/H(+) exchange and may promote Na(+) reabsorption by stabilizing epithelial sodium channel activity.

Autoantibodies in silicosis patients and in silica-exposed individuals

The aim of this study was to evaluate the prevalence of autoantibodies in silica-exposed patients with and without silicosis and without any known rheumatic disease. We studied 61 males exposed to silica for a mean time of 12.2 +/- 10.2 years of exposure. A total of 72.1% (44/61) of them presented with pulmonary silicosis. As control group we included 62 healthy males. In all samples we screened for rheumatoid factor (latex agglutination), antinuclear antibodies (indirect immunofluorescence), anti Scl-70 (ELISA) and ANCA (indirect immunofluorescence technique). One patient (1.6%) of the silica group had weakly positive ANA (titer 1:80, centromeric pattern); one (1.6%) had atypical ANCA and seven patients (11.4%) presented positive rheumatoid factor (values range from 8 to 32 UI/ml). One control patient had a positive RF and none of them had positive ANA or ANCA. All patients and controls were negative for anti-Scl-70. The finding of positive RF was higher in the silica-exposed patients (p = 0.032; Fisher). All patients with positive RF had pulmonary silicosis. In the silica-exposed group we could not find a relationship between the presence of RF and age (p = 0.21; Mann-Whitney), smoking habits (p = 0.25; Fisher) but a positive relationship was found with exposure time to silica dust (p = 0.005; Mann-Whitney). We conclude that there was 11.4% prevalence of low titer RF in the silica-exposed patients without known rheumatic disease. RF was more common in patients with longer exposure to silica dust and appeared only in those with silicosis. The presence of ANA, Scl-70 and ANCA was the same as in the control population.

Antinuclear antibodies in asthma patients- a special asthma phenotype?

Several studies reported the appearance of asthma and autoimmune conditions in the same patient, but the clinical significance of this association was not yet assessed. One hundred asthmatic patients were observed for one year evolution with death, severe exacerbations, intake of > 1000 micrograms of beclometasone or equivalent (high ICS) and FEV1 decline >100 ml, in relation with ANA (ELISA), sputum and blood eosinophilia (EO), NSAID intolerance, BMI >25, chronic rhinosinusitis, smoking status and FEV1 <30% predicted (low FEV1). After 1 year of observation, there were 5 deaths, 28 severe asthma exacerbations requiring hospitalisations, 24 cases requiring high inhaled corticosteroid intake, and 19 patients with fast FEV1 decline (>100 ml/year). Multiple regression analysis pointed out several different independent risk factors for severe asthma evolution: for death presence of ANA (P=0.037), NSAID intolerance (P<0.001) and low FEV1 (P=0.021); for evolution with severe exacerbations ANA (p=0.011), sputum EO (P<0.001), smoking (P=0.044) and NSAID intolerance (P=0.022); for high ICS intake ANA (P=0.036), sputum EO (P=0.026) and low FEV1 (P=0.006); for FEV1 decline >100 ml ANA (P=0.006), sputum EO (P=0.037), BMI>25 (P=0.046) and NSAID intolerance (P=0.017). The presence of ANA is an independent risk factor in asthma for evolution with death, severe exacerbations, high inhaled corticosteroid intake and FEV1 decline >100 ml.

Pre- and postsynaptic effect of galanin on excitatory synaptic transmission in rat spinal dorsal horn neurons

Galanin, a 29 ⁄ 30 amino acid neuropeptide, and three types (GalR1-3) of receptors for this neuropeptide, which are expressed in rat dorsal root ganglion neurons and spinal dorsal horn neurons, play a pivotal role in regulating nociceptive transmission to the spinal dorsal horn from the periphery. Intrathecal administration of galanin in rats modulates nociceptive behavior in a biphasic manner such that galanin at low and high doses produces nociception and antinociception, respectively. In order to know cellular mechanisms for this effect, we investigated the effect of galanin on glutamatergic spontaneous excitatory synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by use of the whole cell patch-clamp technique. In 83% of the neurons examined (n=29), galanin (0.03 µM) superfused for 2 min increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) recorded at -70 mV without a change in its amplitude and also in holding currents. The remaining neurons produced a small outward (n=4) or inward current (n=1). This presynaptic effect was dose-dependent with an EC50 value of 0.0029 µM, and was mimicked by a selective GalR2⁄R3 agonist galanin (2-11) but not a selective GalR1 agonist M617. Superfusing galanin at a higher dose such as 0.1 µM for 2 min produced an outward current in 39% of SG neurons tested (n=33). Twelve % of the SG neurons had an inward current and the remaining neurons did not change holding currents. The outward current produced by galanin had an EC50 value of 0.042 µM, and was mimicked by M617 but not galanin (2-11). The sEPSC frequency increase and outward current persisted in the presence of a Na+-channel blocker tetrodotoxin.It is concluded that galanin at lower doses enhances the spontaneous release of L-glutamate from nerve terminals by activating GalR2⁄R3 while galanin at higher doses produces a membrane hyperpolarization by activating GalR1 in the SG. This pre- and postsynaptic effects could contribute to at least a part of the behavioral effect of galanin.

S4.26 Evidence for the presence of a Na+/H+ antiporter subunit in Rhodothermus marinus complex I

S4.26 Evidence for the presence of a Na+/H+ antiporter subunit in Rhodothermus marinus complex I

Drug‐Induced Lupus

Drug-induced lupus (DIL) is a rare adverse reaction to a large variety of drugs with features resembling those of idiopathic systemic lupus erythematosus (SLE). It usually develops only after months and, quite commonly, years of treatment with the offending agent, although latencies of days or weeks have been described in some instances. There are some indications that the risk of DIL can increase with higher daily and cumulative doses and with longer duration of therapy. There are no definitive and commonly accepted diagnostic criteria for DIL, but the following guidelines have been proposed: (a) sufficient and continuing exposure to a specific drug, (b) at least one symptom compatible with SLE, (c) no history suggestive of SLE before starting the drug, and (d) resolution of symptoms within weeks (sometimes months) after discontinuation of the putative offending agent. In addition, it is frequently suggested that the presence of ANA is required for the diagnosis of DIL. However, negative ANA test results should not automatically preclude such a diagnosis, particularly if a patient has other autoantibodies associated with SLE/DIL.

From the authors

<h3>Background</h3> Biological agents (BA) are high efficacy options for current therapy for patients (pts) with juvenile idiopathic arthritis (JIA). They are successfully used not only for the arthritis but also for uveitis, psoriasis and inflammatory bowel disease (IBD). However, paradoxical induction of these conditions in pts treated with BA is a well-established phenomenon. <h3>Objectives</h3> To evaluate the frequency of new onset of uveitis, psoriasis or IBD occurring under BA therapy in JIA pts, to establish clinical features, which may be associated with development of such effects. <h3>Methods</h3> Retrospective cohort study involved all JIA pts (740) who were treated with BA in our clinic from 2004 to 2016. All cases of new onset (<i>no</i>)-uveitis/psoriasis/IBD collected; clinical features of disease onset and course, activity level, JIA category, exposure to Methotrexate (MTX) and BA, presence of ANA, HLA B27 were studied. <h3>Results</h3> We identified 20 (2.7%) pts (11 female/9 male) with <i>no</i>-uveitis under BA, mostly during etanercept (ETA) therapy (18 cases from 285 ETA courses, 6.3%), 1/112 - in abatacept (ABA) and 1/233 - in adalimumab (ADA); 2.46 events/100 patient-year (PY) vs 0.31 in ABA, and 0.15 in ADA), ETA exposure was 14.9±9.9 months (mo). JIA subtypes were as follows: RF-neg polyarthritis 5 (25%), persistent oligoarthritis 3 (15%), extended oligoarthritis 10 (50%) ERA - 2 (10%). JIA started in this group at the age of 4.5±3.9 yrs.18/20 patients had high laboratory activity (CRP 55±25 mg/l) and severe arthritis before BA initiation. Most of pts (16/20) achieved 90–100% ACRpedi-response by the uveitis development. 1 pt was treated by ADA for the 71 mo <i>and</i> switched to ABA. in 1 pt <i>no</i>-uveitis was obseved under ABA. 12/20 pts were ANA-positive, 10/20 pts had HLAB27, 1 pt did not have ANA or HLAB27. Uveitis was occurred earlier in ANA plus B27 positive pts (mean exposure - 10.7 mo vs 27.4 in ANA+ and 21.6 in B27+ pts). 17/20 (85%) of pts received MTX. In all cases of <i>no</i>-uveitis BA was switched. 5 pts from 740 (0.7%) developed <i>no</i>-psoriasis under BA: INF - 2 cases (0.62/100PY), ADA - 2 (0.15/100PY), ABA -1 (0.31/100PY). JIA subtypes were presented by RF-negative polyarthritis (1), extended oligoarthritis – 3. All received MTX. 1/5 pts was ANA+, 2/5 - HLAB27+. Average age of disease onset was 9.8±7.8 years; BA exposure before psoriasis was 25±11.5 mo. Therapy was continued in 3/5 pts; switched from INF to ADA in 2. Only 1 case of <i>no</i>-IBD was occurred in female patient fulfilled to systemic-onset JIA criteria years. She was treated by INF for the 62 months and switched to ADA due to secondary inefficiency presented by active polyarthritis and sacroiliitis (HLAB27neg), but 2 months later severe gastrointestinal symptoms appeared and diagnosis of Crohn9s disease was established by endoscopy. <h3>Conclusions</h3> Our study suggested that new onset of uveitis, psoriasis and IBD is rare adverse event during BA therapy in JiA. It seemes to be as delayed implication of disease natura. but not therapy complication. Uveitis observed mostly in pts receiving ETA, unlike the psoriasis mainly developed during TNF-monoclonal antibodies using. High activity aggressive manifestations at disease onset and good initial response to BA are typical features for all pts, who developed paradoxical effects under BA therapy. <h3>Disclosure of Interest</h3> None declared

Autoantibody Profile in Systemic Sclerosis as a Marker for Esophageal and Other Organ Involvement in Turkish Populations

Systemic sclerosis (SSc) is a connective tissue disorder of unknown etiology characterized by fibrosis and vascular obliteration in the skin, gastrointestinal tract, lungs, and heart. Our aim was to investigate the autoantibody profile in patients with esophageal involvement of SSc and to describe the relationship between the autoantibody profile and organ involvement in SSc. We studied 47 SSc patients, all with esophageal involvement shown on esophageal manometry. The patients were separated into three groups based on the absence or presence of ANA, Scl70, and ACA. In this study ANA and Scl70 were present more frequently than ACA in patients with esophageal involvement of SSc. Pulmonary involvement and heart involvement were seen more in Scl70-positive and ACA-positive patients, respectively. We conclude that in patients with SSc, closer follow-up with autoantibody profile may enable early diagnosis of specific organ involvement and treatment of debilitating symptoms, with avoidance of potential life-threatening complications.

Contribution of the Na +/Ca 2+ Exchanger to Rapid Ca 2+ Release in Cardiomyocytes

Trigger Ca 2+ is considered to be the Ca 2+ current through the L-type Ca 2+ channel (LTCC) that causes release of Ca 2+ from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current ( I Ca). In this article, we investigate the contribution of the Na +/Ca 2+ exchanger (NCX) to the trigger Ca 2+. Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na +/Ca 2+ exchange delays the Ca 2+ transient by 3–4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na + and Ca 2+ is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na + channel is present in the fuzzy space, the NCX is able to bring enough Ca 2+ into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na + channel and NCX.

Na+ influx triggers bleb formation on inner hair cells

Large blebs form rapidly on apical membranes of sensory inner hair cells (IHCs) when the organ of Corti is freshly isolated from adult guinea pigs. Bleb formation had two distinguishable phases. Initially, we identified small particles labeled with fluorescent annexin V; these rapidly coalesced into larger aggregates. After particle aggregation, a single membrane bleb emerged from cuticular plate at the vestigial kinocilium location, eventually reaching approximately 10 microm maximum spherical diameter; blebs this size often detached from IHCs. Development of blebs was associated with elevated concentration of intracellular Na(+); blocking Na(+) influx through mechanotransduction and ATP channels in the apical pole of IHCs or by replacement of Na(+) with N-methyl-D-glucamine prevented Na(+) loading and bleb formation. Depletion of intracellular ATP, blocking cAMP synthesis, inhibition of vesicular transport with brefeldin A, or inhibition of phosphatidylinositol 3-kinase with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY-294002) significantly reduced bleb formation in the presence of a Na(+) load. Neither the mechanism of blebbing nor the size growth of the IHC blebs was associated with cellular apoptosis or necrosis. Bleb formation was not significantly reduced by disassembling microtubules or decreasing intracellular hydrostatic pressure. Moreover, no polymerized actin was observed in the lumen of blebs. We conclude that IHC bleb formation differs from classic blebbing mechanisms and that IHC blebs arise from imbalance of endocytosis and exocytosis in the apical plasma membrane, linked to Na(+) loading that occurs in vitro.

Ion Transport Across the Gallbladder Epithelium

The function of the gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption. On the contrary, secretions of water and salt take place during the digestive phase. Dysregulation of ion absorption or secretion are common in many gallbladder diseases, such as colelithiasis. Transepithelial absorptions are determined by the Na+/K+ pump on the basolateral membrane, and by several apical membrane Na(+)-coupled transporters. Among these, some isoforms of Na+/H+ and Cl-/HCO3(-) exchangers have been studied. The presence of a Na(+)-Cl(-) simport has been molecularly and functionally characterized in some animal species. The ion transepithelial secretion is mainly dependent on an apical chloride transport attributable to a CFTR-like cAMP-activated channel with high permeability to HCO3(-). The apical membrane electrical potential is one of the factors influencing anion secretion and is maintained by the activity of cAMP-dependent K+ channels. The regulation of the activity of these channels is complex, because of their sensitivity to voltage, and to intracellular calcium and pH. The coordinated interplay underlying the regulation of transporters and channels needs to be clarified yet, as well as the interactions between transporters, channels and aquaporins.

Role of Protein Phosphatase 2A in mGluR5-regulated MEK/ERK Phosphorylation in Neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

Dramatic development of severe SLE in a patient with an incomplete disease

This case report describes the previously-unreported clinical course of a patient with a so-called incomplete systemic lupus erythematosus (SLE), i.e. symptoms related to one organ system only, together with the presence of ANA. He had an indolent course initially and developed, 6 months after the first symptoms, a severe disease with rapid appearance of major and unusual manifestations. The possibility of fast progression and a grave course of an incomplete SLE should be kept in mind. This report is meant to heighten awareness of such an atypical presentation so that prompt and aggressive immunosuppressive therapy may be instituted.

Identification and Characterization of a Na+-Dependent Neutral Amino Acid Transporter, ASCT1, in Rabbit Corneal Epithelial Cell Culture and Rabbit Cornea

Purpose: The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea. Methods: Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34°C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription–polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1. Results: Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 μ moles min−1 mg−1 protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (α -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 × 10−2 μ moles min−1 cm−2), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR. Conclusions: L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.

Homeostasis of Sodium and Potassium Ions in Erythrocytes of the Lamprey Lampetra fluviatilis: Effect of Ion Transport and Metabolic Blockers, and Ionophores

After incubation of lamprey Lampetra fluviatilis erythrocytes in the standard medium for 90–120 min, intracellular Na+ and K+ content remained unchanged (28.7 ± 1.1 and 66.3 ± 1.5 mmol/l cells, respectively, n = 33). The erythrocyte ion content also did not change after treatment of the cells with ion transport inhibitors, Ba2+ and amiloride. Addition of 0.1 mM ouabain to the incubation medium led to a decrease of K+ content by 8.4 ± 1.2 and to an increase of Na+ content by 2.4 ± 0.8 mmol/l/2 h. Similar reciprocal changes in the cellular ion composition were observed after treatment of the erythrocytes by oxidative metabolism inhibitors (rotenone and CCCP—carbonyl cyanide m-chlorophenyl-hydrazone). The metabolic blockers produced more significant ion composition changes in comparison with ouabain. An increase of intracellular Na+ content under effect of CCCP was completely inhibited by amiloride. It can be suggested that inhibition of oxidative metabolism is accompanied by a cell acidification and Na+/H+ exchange activation. Erythrocyte acidification by a K+/H+ ionophore led to a rapid cellular Na+ accumulation, which indicates the presence of a Na+/H+ exchanger with high activity. The K+ ionophore valinomycin produced a relatively small K+ loss from the lamprey erythrocytes to indicate a low anion conductance of the cells. The data obtained indicate an important role of oxidative metabolism in the monovalent ion homeostasis in the lamprey red blood cells.

Arachidonic acid-activated Na +-dependent Mg 2+ efflux in rat renal epithelial cells

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg 2+ deficiency. We examined whether the extracellular Mg 2+ concentration affects AA production and whether AA regulates a putative Na +-dependent Mg 2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg 2+-containing medium for 2 days because they enable us to detect Na +-stimulated Mg 2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg 2+ increased AA release both in the absence and presence of extracellular Na +. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 μM), an inhibitor of cytosolic phospholipase A 2 (cPLA 2) and Ca 2+-independent phospholipase A 2 (iPLA 2), and bromoenol lactone (BEL, 10 μM), an inhibitor of iPLA 2. However, LY-311727 (10 μM), a secretory phospholipase A 2 (sPLA 2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA 2 and iPLA 2 mRNAs, but not sPLA 2. In the mag-fura 2 fluorescence measurements, extracellular Mg 2+ removal caused slight decrease in the intracellular free Mg 2+ concentration ([Mg 2+] i) in the Na +-free condition. The addition of Na + caused a rapid decrease in [Mg 2+] i, indicating the presence of a Na +-dependent Mg 2+ efflux pathway. The Na +-dependent [Mg 2+] i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 μM), indomethacin (10 μM) and 17-octadecynoic acid (ODYA) (10 μM), enhanced the Na +-dependent [Mg 2+] i decrease. Furthermore, the addition of exogenous AA (30 μM) enhanced the Na +-dependent [Mg 2+] i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na +/Mg 2+-exchanger inhibitor. These results suggest that extracellular Mg 2+ removal elevates AA release mediated mainly by iPLA 2 and that AA upregulates the Na +-dependent Mg 2+ efflux in NRK-52E cells.

Sodium transport across the chorioallantoic membrane of porcine placenta involves the epithelial sodium channel (ENaC).

The properties of chorioallantoic membrane derived from Large White Landrace sows at 45, 65 and 100 days gestation are examined. Under short circuit conditions positive charge flows from fetal to maternal sides of the tissue. Na+ is shown to be the sole charge carrier as the short circuit current is inhibited reversibly by fetal applications of amiloride and replacement of Na+ by choline in the Ringer solution, and irreversibly by both fetal and maternal applications of ouabain. The initial short circuit current is smaller at day 100 compared to days 45 and 65. The dose responses to amiloride indicate that the epithelial sodium channel (ENaC) is involved in the movement of Na+ and that it is accessible on the fetal side of the tissue only. Immunostaining shows that the ENaC-alpha subunit is present in both the allantoic membrane and the trophoblast. Uptake studies using microvillous (apical) membrane vesicles suggest it is either inactive or only weakly active at this site. The trophoblast at day 100 has a higher content of ENaC than at days 45 and 65. This is the first report of the presence of ENaC in placental tissues. The effects of ouabain indicate the presence of a Na+ pump that is more readily inhibited by applications of the drug on the maternal aspect of the tissue than on the fetal side. Differential mechanisms may be present that would allow net movement of Na+ in either direction across the chorioallantoic membrane according to the changing demands of the developing fetus.